We study the mechanism and regulation of protein synthesis in eukaryotic cells focusing on regulation by GTP-binding (G) proteins and protein phosphorylation. The first step of protein synthesis is binding the initiator Met-tRNA to the small ribosomal subunit by the factor eIF2. The eIF2 is a GTP-binding protein and during the course of translation initiation the GTP is hydrolyzed to GDP. The eIF2 is released from the ribosome in complex with GDP and requires the guanine-nucleotide exchange factor eIF2B to convert eIF2-GDP to eIF2-GTP. This exchange reaction is regulated by a family of kinases that specifically phosphorylate the alpha subunit of eIF2 on serine at residue 51, and thereby covert eIF2 into an inhibitor of eIF2B. Among the family of eIF2alpha kinases are GCN2 (activated under conditions of amino acid starvation), PKR (activated by double-stranded RNA and downregulates protein synthesis in virally infected cells), PERK (activated under conditions of ER stress), and HRI (activated under conditions of low heme). The factor eIF2 is composed of three polypeptide chains. The gamma subunit of eIF2 is a GTPase that resembles elongation factor EF-Tu. However, in contrast to EF-Tu, which binds tRNAs to the A-site of the 70S ribosome, eIF2 binds Met-tRNAi to the P-site of the 40S subunit. To gain insights into how eIF2 binds Met-tRNAi and then associates with the 40S ribosome, we used directed hydroxyl radical probing to identify eIF2 contacts within the 40S-eIF1A-eIF2-GTP-Met-tRNAi-mRNA (48S) complex. Based on our results, we generated a model of the 48S complex in which domain III of eIF2gamma binds near 18S rRNA helix h44 and eIF2gammaMet-tRNAi contacts are restricted to the acceptor stem of the tRNA. In this model of the eIF2 ternary complex, the Met-tRNAi is rotated nearly 180 degrees relative to the position of the tRNA in the EF-Tu ternary complex. Thus, despite their structural similarity, eIF2 and EF-Tu bind tRNA in substantially different manners, and we propose that the tRNA-binding domain III of EF-Tu has acquired a new function in eIF2gamma to bind the ribosome. Whereas protein synthesis is known to play a critical role in learning and memory in diverse model systems, human intellectual disability syndromes have not been directly associated with alterations in protein synthesis. Our collaborators in Israel and Germany identified a human X-chromosomal neurological disorder characterized by intellectual disability and microcephaly. Mapping studies identified the causative mutation as a single base change resulting in a missense mutation in eIF2gamma (encoded by EIF2S3). Biochemical studies of human cells overexpressing the eIF2gamma mutant and of yeast eIF2gamma with the analogous mutation revealed a defect in binding the eIF2beta subunit to eIF2gamma. Consistent with this loss of eIF2 integrity, the mutation in yeast eIF2gamma impaired translation start codon selection and eIF2 function in vivo in a manner that was suppressed by overexpression of eIF2beta. These findings directly link intellectual disability to impaired translation initiation, and provide a mechanistic basis for the human disease due to partial loss of eIF2 function. In the past year, additional collaborators have identified another mutation in eIF2gamma that causes intellectual disability, and we are the process of characterizing this new mutant. Previously, we demonstrated that, when expressed in yeast, human PKR phosphorylates the alpha subunit of eIF2 on Ser51 resulting in inhibition of protein synthesis and yeast cell growth. In order to subvert the anti-viral defense mediated by PKR, viruses produce inhibitors of the kinase. Poxviruses express two different types of PKR inhibitors: a pseudosubstrate inhibitor (e.g. vaccinia virus K3L protein that resembles eIF2alpha) and a double-stranded RNA binding protein called E3L. Expression of vaccinia virus K3L or E3L protein or the related variola (smallpox) virus C3L or E3L protein, respectively, restored the growth of yeast expressing PKR. We are currently characterizing mutations in PKR that confer resistance to E3L inhibition, and studying the functional domains of the E3L protein. The E3L protein contains an N-terminal Z-DNA binding (Zalpha) domain and a C-terminal dsRNA-binding domain (dsRBD). While E3L is thought to inhibit PKR activation by sequestering dsRNA activators and by directly binding the kinase, the role of the Zalpha domain in PKR inhibition is unclear. We recently showed that the E3L Zalpha domain is required to suppress the growth-inhibitory properties associated with expression of human PKR in yeast, to inhibit PKR kinase activity in vitro, and to reverse the inhibitory effects of PKR on reporter gene expression in mammalian cells treated with dsRNA. Whereas previous studies revealed that the Z-DNA binding activity of E3L is critical for viral pathogenesis, we identified point mutations in E3L that functionally uncoupled Z-DNA binding and PKR inhibition. Thus, our studies demonstrate that the variola virus E3L Zalpha domain, but not its Z-DNA binding activity, is required for PKR inhibition, and they support the notion that E3L contributes to viral pathogenesis by targeting PKR and other components of the cellular anti-viral defense pathway. Whereas the protein kinases GCN2, HRI, PKR, and PERK specifically phosphorylate eIF2alpha on Ser51 to regulate global and gene-specific mRNA translation, eIF2alpha is dephosphorylated by the broadly acting serine/threonine protein phosphatase 1 (PP1). In mammalian cells, the regulatory subunits GADD34 and CReP target PP1 to dephosphorylate eIF2alpha; however, as there are no homologs of these targeting subunits in yeast, it was unclear how GLC7, the functional homolog of PP1 in yeast, is recruited to dephosphorylate eIF2alpha. We showed that a novel N-terminal extension on yeast eIF2gamma binds to GLC7 and targets it to dephosphorylate eIF2alpha. Truncation or point mutations designed to eliminate the PP1-binding motif in eIF2gamma impaired eIF2alpha dephosphorylation both in vivo and in vitro. Moreover, replacement of the N terminus of eIF2gamma with the GLC7-binding domain from GAC1 or fusion of heterologous dimerization domains to eIF2&#947; and GLC7, respectively, maintained eIF2alpha phosphorylation at basal levels. Taken together, our results indicate that, in contrast to the paradigm of distinct PP1-targeting or regulatory subunits, the unique N terminus of yeast eIF2gamma functions in cis to target GLC7 to dephosphorylate eIF2alpha. We are also studying the translation factor eIF5A. eIF5A is the sole protein containing the unusual amino acid hypusine N-epsilon-(4-amino-2-hydroxybutyl)lysine. This hypusine residue in eIF5A is found in all eukaryotes and archaea. Using molecular genetic and biochemical studies, we showed that eIF5A promotes translation elongation, and this activity is dependent on the hypusine modification. As eIF5A is a structural homolog of the bacterial protein EF-P, we proposed that eIF5A/EF-P is a universally conserved translation elongation factor. More recently, we showed that eIF5A in yeast, like bacterial EF-P, stimulates the synthesis of proteins containing runs of consecutive proline residues. Consistent with these in vivo findings, we found that synthesis of polyproline peptides in reconstituted yeast in vitro translation assays was critically dependent on eIF5A. Finally, directed hydroxyl radical probing experiments localized eIF5A binding near the E site of the ribosome with the hypusine residue of eIF5A adjacent to the acceptor stem of the P-site tRNA. Thus, we propose that eIF5A, like its bacterial ortholog EF-P, stimulates the peptidyl-transferase activity of the ribosomes and facilitates the reactivity of poor substrates like proline.